A cell is the fundamental building block of tissue and receives and generates signals directing its growth and division, differentiation, and death. Its interactive communication with other cells is the basis for tissue and organ formation. Our understanding of the complexities of these processes would greatly benefit from improved abilities to analyze the molecular and biochemical processes of single cells. The objective of this proposed study is to develop and characterize a novel assay platform for the real-time monitoring of proteins secreted by single cells. The platform consists of a microfabricated chip with micron-sized wells to corral single cells and functionalized cantilever sensing elements. In this phase of the project, we will use fluorescence as the readout method for detecting with bound antigen. In the next phase, we plan to develop a label-free readout mechanism utilizing measurable changes in sensing cantilever dynamics as target proteins become bound to the cantilevers. The Specific Aims of this application are: Aim 1, to design and fabricate Cell-Well chips with microcantilevers positioned at the well periphery; Aim 2, to construct and characterize functional Cell- Well biosensors; Aim 3, to utilize the Cell-Well biosensors to detect proteins secreted by a few cells and by single cells; Aim 4, to utilize the Cell-Well biosensors to monitor changes in protein secretion into cell media as a function of cell stimulation. To achieve these Aims, we will use silicon micromachining technology to construct the Cell-Well chips and Nano eNabler technology to functionalize the microcantilevers. Micron-sized capture domains offer many distinct advantages to this study including accuracy of loading cantilevers, increased sensitivity due to} analyze harvesting}, improved binding kinetics, smaller reaction volume, potentially reduced reaction times and reduced amounts of analyze and ligand in assays. Also, by constraining the cells and their secreted proteins to very small volumes (<500 pl), higher detection sensitivities, in terms of protein mass, may be expected. Therefore, the Cell-Well chip is expected to provide a realistic platform for the study and detection of proteins released by a few or even single cells. Cells, the fundamental building block of tissues, are biochemically heterogeneous. The ability to analyze the molecular and biochemical processes at the single cell level would provide new insights into the overall understanding of many biological processes. The objective of this proposed work is to develop and characterize a novel assay platform for monitoring of proteins secreted by a single cell. [unreadable] [unreadable] [unreadable]